Proteins are essential for many cell functions including signaling, activation, generation of mechanical energy, and processing of biochemical reactions. One major class of proteins is that of enzymes: molecular machines that work in cycles to facilitate rapid progression of chemical reactions. Computational studies and simulations are likely to shed light on the operation of these biologically important molecules. However, actions of enzymes are conducted on extended spatial and temporal scales that are a challenge for computational studies. Theories and algorithms are developed in my group to address these challenges. These technologies include algorithms for reaction path calculations and the theory of Milestoning. On the application side, the focus of the next grant cycle is on HIV RT. We will investigate critical steps in the machine cycle of the enzyme HIV RT, which include the weak binding of the nucleotide to the protein surface, the chemical step, byproduct release, and sliding of the protein along the DNA to free space for a new substrate. On the computational and theoretical side, we are planning three advances. In the first advance, we will introduce a novel algorithm for the calculations of minimum free energy pathways. There are two advantages to the new algorithm that we call ?chain growth?. The first is that it works in a large space of coarse variables. The second advantage is that the algorithm does not require an initial guess for the path, and, therefore, the optimization is significantly easier than other algorithms that we have used in the past. The second advance planned is the development of an automated script that will run the Milestoning algorithm using essentially any Molecular Dynamics software package. The MD packages will generate the short trajectories required in the Milestoning analyses, and the script will provide the initial conditions, analyze the short trajectories for convergence, and initiate new trajectories as needed. If the simulation is converged the script will compute kinetic and thermodynamic observables. The script will make it easier for researchers to use the Milestoning theory and algorithm within the framework of their familiar MD program. The third advance will compute an optimal reaction coordinate (iso-committor surfaces) from Milestoning trajectories and will make it possible to further refine, analyze, and test the simulations.